Bioscience Repo s, Vol. 7, No. 6, 1987
E ec o
E. Coli
Endo oxin on Tempe a u e,
Oxygen Consump ion and B own Adipose
Tissue The mogenesis in Ra s and Mice
G. Jennings 1 and M. Elia
Recei ed June 3, 1987
KEY WORDS: endo oxin; b own adipose issue; ene gy expendi u e.
The e ec s o E. coli endo oxin 0127 B8 on oxygen consump ion, empe a u e, and on
he ac i i y o he p o on conduc ance pa hway in b own adipose issue (BAT) we e
in es iga ed in a s and mice. In a s an inc ease was obse ed in ec al and skin
empe a u e, whole body oxygen consump ion and GDP binding in BAT. In mice only
he ise in ec al and skin empe a u e we e signi ican ly changed by endo oxin
adminis a ion.
These indings sugges ha in some species BAT is in ol ed in he p oduc ion o
endo oxin induced e e and inc eased ene gy expendi u e.
INTRODUCTION
Fe e and inc eased me abolic a e commonly occu a e inju y, i espec i e o
whe he he inju y is acciden al, su gical, he mal o sep ic (1). The mechanisms ha
p oduce hese esponses a e no en i ely unde s ood al hough in lamma o y media o s
and a numbe o s ess ho mones, including ca echolamines, glucagon and
co icos e oids a e p obably in ol ed (2). In small animals endo oxin induced e e
may also be due o conse a ion o hea by he ail (3), and o inc eased hea
p oduc ion by b own adipose issue (BAT) (4, 5). This las sugges ion has been based
on wo pieces o indi ec e idence: (a) epo s ha endo oxin inc eases blood low o
b own adipose issue (5, 6), and (b) he associa ion be ween an inc ease in blood low
h ough BAT and non-shi e ing he mogenesis (ac i a ion o BAT he mogenesis) in
Dunn Nu i ion Labo a o y, Downhams Lane, Mil on Road, Camb idge.
1 To whom co espondence should be add essed.
517
0144-8463/87/0600-0517505.00/0 9 1987 Plenum Publishing Co po a ion
518 Jennings and Elia
animals exposed o he c01d and gi en no ad enaline (7-10). In his s udy we epo he
esul s o he e ec s o endo oxin adminis a ion on he ac i i y o he BAT
mi ochond ial p o on conduc ance pa hway (GDP binding), which plays a
undamen al ole in egula o y he mogenesis in small animals (11-14). The esul s a e
ela ed o changes in whole body oxygen consump ion and o changes in ec al, skin
and ail empe a u es.
METHODS AND MATERIALS
Animals and Expe imen al P ocedu e
Expe imen s we e ca ied ou on 3-4 mon h old male mice o he As on a ie y,
and male Dunn a s (app ox. 300 g) o he No wegian hooded a ie y. Bo h g oups o
animals we e main ained on a 12 h ligh /12 h da k cycle (ligh pe iod om 7.00 a.m.).
Animals we e acclima ed a a ious empe a u es o a leas 3 weeks be o e use.
All expe imen al animals we e gi en a single in ape i oneal injec ion (3 mg/kg) o
E. coli
endo oxin ype 0127 B8 (bu anol ex ac ed) which was ob ained om Sigma
Chemical Co. L d., Poole, Do se , UK. The endo oxin was dissol ed in s e ile saline
(0.9 ~ w/ ) a a concen a ion o 1 mg/ml o mice and 2 mg/ml o a s. The con ol
animals we e gi en saline wi hou endo oxin.
0 2 Consump ion
The e ec o endo oxin on 0 2 consump ion was assessed in mice acclima ed a
he moneu al empe a u e (32~ o 3 weeks.
P elimina y s udies had shown ha such animals esponded o endo oxin
injec ion by p oducing a mo e p onounced and ep oducible ise in ec al empe a u e
(compa ed o saline con ols) han animals housed a 22~ The e ec o endo oxin on
oxygen consump ion was also assessed in mice ha we e acclima ed a 22~ o 3
weeks and ans e ed o he moneu al empe a u e o 24 h .
The a s we e acclima ed a 26~ 2~ (lowe end o hei he moneu al
empe a u e), which was shown in p elimina y s udies o be sui able o elici ing an
endo oxin induced e e . Whole body oxygen consump ion was measu ed
con inuously o six hou s a e endo oxin adminis a ion. The measu emen s we e
made a he he moneu al empe a u e o each animal (32~ o mice and 28~ o
a s) using a closed ci cui oxygen consump ion sys em (15).
Tempe a u e Measu emen
The e ec o endo oxin on empe a u e was assessed in he ollowing g oups o
animals: (a) a s acclima ed a 26 +_ 2~ (b) mice acclima ed a 32~ (c) mice
acclima ed a 22~ and (d) mice acclima ed a 22~ and ans e ed o he moneu al
empe a u e o 24 h .
The measu emen s we e made a he same empe a u e as ha used o acclima ise
he animals (excep animals in g oup (d) which we e es ed a 32~
Rec al empe a u e was only measu ed in g oups (c) (n = 6) and (d) (n = 6), while
ec al, skin and ail empe a u es we e measu ed in g oups (a) (n = 12) and (b) (n = 9).
Endo oxin and B own Fa The mogenesis
519
Rec al, skin and ail empe a u es we e measu ed using he mocouples connec ed
o a digi al display he mome e . Skin measu emen s we e ob ained by placing he
he mocouple benea h he u on he ea lank o he animal. Tail empe a u es we e
aken a a poin 2 cm om he base o he ail. Du ing hese measu emen s, he
he mocouples we e insula ed wi h co on wool and a polys y ene co e . The
insula ion was su icien o gi e a cons an eading (i.e. o p e en any d i in he
eadings due o exchange o hea wi h he en i onmen ). Rec al empe a u es we e
aken by inse ing a p obe o a dep h o 1.5 cm o mice and 5 cm o a s.
B own Adipose Tissue
In his se o expe imen s he e ec o endo oxin on he ac i i y o he p o on
conduc ance pa hway o in e scapula BAT was s udied. The animals we e killed by
ce ical disloca ion 4 hou s a e endo oxin adminis a ion. In e scapula BAT was
apidly emo ed, cleaned o any adhe ing whi e adipose issue o connec i e issue,
and weighed. The b own a was hen homogenized in a medium (pH 7.2) con aining
250mM suc ose, 200#M po assium EDTA and 1 mM HEPES. Samples o he
homogena e we e aken o he measu emen o cy och ome oxidase ac i i y (EC
1.9.3.1) a 25~ by a spec opho ome ic me hod (16), and o he measu emen o
o al issue p o ein con en (17).
The majo po ion o he BAT homogena e was used o p epa e mi ochond ia
(18), which we e subsequen ly assayed o GDP binding (18), and he concen a ion o
he mi ochond ial uncoupling p o ein by a modi ica ion o a solid phase
adioimmunoassay (19), as desc ibed p e iously (20). A abbi an i- a uncoupling
p o ein se um was used in he adioimmunoassay, oge he wi h a a p o ein
s anda d.
S a is ical analysis o g oup da a was pe o med using he s uden s - es .
RESULTS
02 Consump ion
Endo oxin p oduced a small non-signi ican ise in 02 consump ion in bo h he
mice acclima ed a 32~ (10 ~ inc ease abo e con ols) and also in hose acclima ed a
22~ ans e ed o 32~ o 24 h be o e he expe imen (13~ inc ease abo e
con ols).
In he a s 02 consump ion was signi ican ly ele a ed om 3 h a e endo oxin
ea men (see Fig. 1). Be ween 34 h he measu emen s we e 19 ~ highe (p < 0.01)
han in con ol animals and be ween 4-5 h hey we e 21 ~ highe (p < 0.05). The
ini ial measu emen s in bo h saline and endo oxin injec ed animals we e high as a
esul o he injec ion and he change in hei en i onmen .
Tempe a u e Measu emen s
Ra s (acclima ed a 26~ and mice (acclima ed a 32~ exhibi ed highe ec al
empe a u es a e endo oxin adminis a ion han a e saline. A maximum di e ence
o 1.4~ was obse ed in a s a 4 h , and 0.9~ in mice a 4.5 h. These di e ences we e
520 Jennings and Ella
.-~
_
10
O
JO
o=20 _
e,-
E16
_
X
0
=12
_
0-1
T
1-2 2-3
o
i
!
i
3-4 4-5
Hou s:Pos - endo oxin
5-6
Fig. 1. Change in es ing me abolic a e a e saline ([]) o endo oxin (I)
adminis a ion in a s. *p < 0.05; **p < 0.01 compa ed wi h saline ea ed
a s. 6 a s pe g oup, esul s a e mean alues o e one hou pe iods +__ SEM.
40.6
39,8
39.0
38.2
39.0
ou
9 38.2
37.4
6 36.6
~o
P
31.8
3 .0
30,2
29.4
(a)
38.4
9 O0 ~O~
: J 38.6
Rec al 37.8
1~ 370
(n-
-
38.6
Skin O~O II ~ 37.8
a
37.0
~ 36.2
~- 34,6
33.8
Tail 33.0
32.2
31.8
30.8
!
i i-- i
0 2 4 8
(b)
Rec al
(n=9)
Skin
(n=81
Tail
O 2 4 6 8
Hou s:Pos -endo oxin Hou s:Pos -endo oxln
Fig. 2. E ec o endo oxin on ec al, skin and ail empe a u e in a s (Fig. 2a) and mice (Fig. 2b). (0)
Endo oxin ea ed animals. ((3) Saline con ols. Resul s a e means _+ SEM wi h he numbe o animals/
g oup in pa en heses. *p < 0.05; **p < 0.01; ***p < 0.001 signi ican di e ences be ween endo oxin and
saline injec ed animals a e indica ed.
Endo oxin and B own Fa The mogenesis 521
also e lec ed in skin empe a u es which, a hei peak, we e 0.8-0.9~ g ea e in
endo oxin injec ed animals han saline injec ed animals. Howe e , in nei he a s no
mice was he e an inc ease in ail empe a u e (see Fig. 2).
In mice acclima ed a 22~ endo oxin adminis a ion caused a maximum
inc ease in mean ec al empe a u e which was only 0.4~ abo e con ol animals
CN.S.). The mice ha we e acclima ed a 22~ and ans e ed o 32~ 24 h be o e
endo oxin adminis a ion esponded by p oducing a la ge ise in ec al empe a u e
(0.7~ - p < 0.02).
B own Adipose Tissue Measu emen s
The weigh o BAT and GDP binding o BAT mi ochond ia in mice we e no
signi ican ly a ec ed by endo oxin adminis a ion (Table 1). In con as injec ion o
endo oxin o he a caused a signi ican dec ease in he weigh o in e scapula BAT
(18.5 %) and an inc ease in GDP binding (67 %). These changes we e no associa ed
wi h signi ican al e a ions in o al p o ein, cy och ome oxidase ac i i y o
concen a ion o uncoupling p o ein in BAT (see Table 2).
Table 1. E ec o E. coli endo oxin 0127 B8 (bu anol ex ac ) on b own
adipose issue in mice
Con ol Endo oxin
Acclima ised a 32~
BAT w . (mg) 179 • 8 163 • 12
GDP bound 30 +_ 6 43 +_ 9
~mol/mg mi o. p o .)
(n = 6 pe g oup)
Acclima ised a 22~ h a 32~
BAT w . (nag) 73 _+ 7 75 ___ 7
GDP bound 86 _+ 18 102 • 15
~mol/mg mi o. p o .)
(n --- 5 pe g oup)
BAT was ob ained 4 hou s pos endo oxin adminis a ion.
Resul s a e mean • SEM.
DISCUSSION
This s udy demons a es h ee aspec s o he esponse o animals o endo oxin.
The i s ela es o an appa en species di e ence in esponse o he same endo oxin.
Thus, in he a ec al empe a u e, me abolic a e and b own a he mogenic indices
all changed signi ican ly a e endo oxin adminis a ion, whe eas in he mouse, despi e
simila ends o hose ound in he a , signi ican changes in empe a u e could only
be demons a ed wi h espec o empe a u e (and hese only occu ed a
he moneu al empe a u e). This sugges s ha he a may be a be e model han he
mouse, o s udying he hype me abolic esponse o inju y.
522
Table 2.
Jennings and Ella
E ec o E. coli endo oxin 0127 B8 (bu anol ex ac ) on b own
adipose issue in a s
Con ol Endo oxin
BAT w . (mg) 430+ 21 355 + 18"
GDP bound 182 ___ 32 304 + 28**
~mol/mg mi o. p o .)
Cy o. oxid. ac i i y 83.9 + 8.0 93.0 + 10.2
~mol cy o. c oxid./min.)
Uncoupling p o ein 24.6 ___ 2.0 31.8 + 3.2
~ug/mg mi o. p o ein)
To al BAT p o ein (nag) 23.3 _ 1.0 23.2 + 1.2
BAT was ob ained 4 hou s pos endo oxin adminis a ion (n = 6 in each
g oup).
Resul s a e mean + SEM.
Signi ican di e ences be ween endo oxin and saline injec ed animals a e
indica ed: *p < 0.05, **p < 0.02.
The second poin ela es o ail empe a u e. A ise in he empe a u e o he blood
pe using he ail almos ce ainly occu ed a e endo oxin adminis a ion because
he e was a ise in ec al (co e) empe a u e. Al hough his migh be expec ed o
p oduce a ise in ail empe a u e, no such ise occu ed. The e o e i is easonable o
conclude ha endo oxin educed he pe usion o he ail and he p opo ion o o al
hea los ia he ail. Such conclusions a e consis en wi h he haemodynamic changes
obse ed in he ail o he a a e in a enous injec ion o a di e en ype o endo oxin
(E. coli 0111) (3).
Thi dly, and mos impo an ly, he s udy has p o ided e idence ha in he a ,
adminis a ion o E. coli endo oxin ac i a es b own adipose issue. This is appa en
om he loss o BAT weigh , which is p esumably associa ed wi h inc eased lipolysis
and a oxida ion, and he inc ease in GDP binding in BAT mi ochond ia (due o
unmasking o binding si es) which e lec s ac i a ion o he p o on conduc ance
pa hway. Cy och ome oxidase ac i i y and he concen a ion o b own a uncoupling
p o ein, as expec ed, did no change du ing he cou se o his sho s udy. The da a
he e o e indica e ha unde he ci cums ances o s udy, a specialised he mogenic
issue becomes ac i a ed by E. coli endo oxin ype 0127:B8 and con ibu es o he
py exia and inc eased ene gy expendi u e. The py exia i sel may also inc ease ene gy
expendi u e by inc easing he o e all a e o me abolic p ocesses in he a ious issues
o he animal.
The mice acclima ised a 32~ had a ma kedly dep essed BAT ac i i y, which may
ha e es ic ed eac i a ion o he p o on conduc ance pa hway in esponse o
endo oxin. Howe e his should no ha e been a p oblem in he mice ha we e
main ained a 22~ and exposed o 32~ o only 24 h p io o endo oxin
adminis a ion. Since his g oup o mice also showed no inc ease in GDP binding in i s
BAT mi ochond ia, i is sugges ed ha mouse BAT, unde he condi ions o s udy, is
no ac i a ed by E. coli endo oxin ype 0127:B8.
Whe he b own a becomes ac i a ed a e o he o ms o auma and a e
adminis a ion o o he ypes o endo oxins emains o be elucida ed.
Endo oxin and B own Fa1 The mogenesis
523
ACKNOWLEDGEMENTS
We wish o hank D P. Lunn o help ul discussions and Miss M. Howle and
M s A. Flack o assis ance in he animal house and Miss D. Fo dham o yping he
manusc ip .
REFERENCES
1. E ia, M. (1982). Clinical Nu i ion 1:173-192.
2. Wa e s, J. M., Bessey, P. Q., Dina eIlo, C., Wol , J. M. and Wilmo e, D~ W. (1986). A chi es Su ge y
121:179-190.
3. Szeke y, M. and Szelemyi, Z. (1979). Ac a Physiol. Acad. Sci. Hun 9. 53:265-277.
4. Szekely, M., Szelemyi, Z. and Sumeyi, I. ( 973). Ac a PhysioL Aead. Sei. Hung. 43:85-88.
5. Ha is, W. H., Fos e , D. O. and Nadeau, B. E. (1985). Can. J. Physiol. Pha macol. 63:595-598.
6. Jepson, M. M., Cox, M., Ba es, P. C., Ro hwell, N. J., S ock, M. J, Cady, E. B. and Millwa d, D, J.
(1987). Am. J. Physiol. 252: (Endoc inol. Me ab. 15) E581-E587.
7. Helm, T. and Hull, D. (1966). J. Physiol. (London) 186:42-55.
8. Ja ai, I. (I969). J. Physiol. (London) 202:559-567.
9. Fos e , D. O. and F ydman, M. L (1979). Can. J. Physiol. Pha macol. 57:257-270.
I0. Thulby, P. L. and T ayhu n, P. (1980). P luoe s A ch. 385:193-201.
I . Nicholls, D. G. and Locke, R. M. (1984). Physiol. Re . 64:1-64.
12. Ro hweI1, N. J. and S ock, M. J. (I984). Recen Ad . Physiol. 11:349-382.
13~ Himms-Hagen, J. (1985). Ann~ Rew Nu . 5:69-94.
14. Cannon, B. and Nede gaa d, J. (1985). Essays Biochem. 20:110-164.
15. S ock, M. J. (1979). J. App. Physiol. 39:849-850.
16. Yane ani, T. and Ray, G. S. (1965). J. Biol. Chem. 240:3392--3398.
7. Schac e le, G. R. and Pollack, R. L. (1973). Ana y . Biochem. 51:654-655.
8. Goodbody, A. E. and T ayhu n, P. (1981). Biochem. J. 194: 019-1022.
19. Lean, M. E. J., B anch, W. J., James, W. P. T., Jennings, G. and Ashwell, M. (1983). Biosci. Rep, 3:61-
71.
20. Lean, M. E. J., James, W. P. T., Jennings, G. and T ayhu n, P. (1986). Clin. Sci. 71:291-297.
